Techniques for assaying a liquid sample for the presence and/or concentration of specific substances are known to those skilled in the art. Examples of such techniques include radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA), and related protein-binding methodologies. All of these techniques involve the binding of a compound to some sort of specific receptor and accordingly fall into the general category of ligand/anti-ligand assays, a category not limited to any special type of interaction occurring in the assay or to any particular type of components participating in the reaction. All ligand/anti-ligand assays are based on two premises: (1) that certain pairs of substances (the ligand and the anti-ligand) have a strong and specific affinity for each other, that is, they will tend to bind to each other, while binding little or not at all to other substances; and (2) that methods can be developed that allow detection of ligand/anti-ligand binding interactions once complexes have formed. As used herein, ligand is defined as the substance to be detected, and anti-ligand the substance used to probe for the presence of the ligand. In some ligand/anti-ligand assays, an additional, perhaps modified, ligand may be used that competes with the substance to be detected for binding sites on the anti-ligand. Immobilization of either the ligand or anti-ligand will in some cases facilitate detection of the reaction product.
Ligand/anti-ligand reactions can be detected by a variety of methods using various markers to label the reaction product. Currently, the most commonly used markers include enzymes and fluorochromes and radioactive compounds.
Ligand/anti-ligand assay methods can be applied to enzyme immunoassays to detect the presence or absence of antibodies or antigens, i.e., ligands in a sample. In recent years, the use of EIAs and ELISAs has become increasingly important for the detection and quantitation of biologically active substances. In typical ELISA methodology, antigens can be detected directly or indirectly by capture of the antigen with a specific antibody and detection of the bound antigen by use of enzyme-labeled antibodies which catalyze, under appropriate conditions, a reaction with a substrate. The enzyme activity is generally detected by formation of a colored reaction product. Several enzymes, including alkaline phosphatase, horseradish peroxidase and glucose oxidase have been coupled to antigen or antibody. Horseradish peroxidase (HRP) is commonly used. Several substrates are available for (HRP). For visual detection, the substrate will usually comprise a solution of hydrogen peroxide and a chromogenic material, such as, o-phenylenediamine or tetramethylbenzidine, which manifests a color upon oxidation.
In fluorescent immunoassay techniques (FIA) antigens can be labeled directly or indirectly with fluorochrome-labeled antibodies. Fluorochromes are dyes that absorb radiation (e.g., ultraviolet light), are excited by it, and emit light (e.g., visible light). The most commonly used fluorochromes are fluorescein isothiocyante and tetramethylrhodamine isothiocyanate.
The detection via ligand/anti-ligand reactions of analytes in a biological sample has been achieved through the use of methods employing radioactively labeled compounds. Various RIA techniques, for the direct or indirect measurement of ligands, are well known to those skilled in the art.
Ligand/anti-ligand assays have applications in the field of medicine, for example, in the diagnosis of infectious diseases. Accurate diagnosis and treatment of infection is possible only after the exact identity of the etiological agent has been established. In the case of microorganisms, species or group specific antigens, i.e., ligands, which may be used to identify the microorganism and/or to further identify its group, can be extracted from, e.g., the whole cell, the cell wall, or cell fragments. It is desirable that assays for the detection of such ligands be simple and fast.
Group A Streptococcal antigen, a polysaccharide, can be advantageously assayed by use of ligand/anti-ligand assays. Beta-hemolytic, pathogenic Group A streptococci are the most common bacterial agent associated with infections of the upper respiratory tract and of the skin in humans. The highest occurrence is typically found in children. Antibiotic therapy is the treatment of choice. If left untreated, a Group A streptococcal infection may lead to more serious complications such as rheumatic fever (Wannamaker, L. E.: Reviews of Inf. Dis., 1:967-973, 1979; Catanzaro, F. J., et al.: Amer. J. Med., 17:749-756, 1954). Because of the high frequency of Group A Streptococci as etiological agents of human disease, simple and fast methods of diagnosis are being sought.
Methods currently used to identify Group A streptococci isolated from culture include bacitracin susceptibility and latex slide agglutination. Confirmatory methods include immunofluorescence, precipitin tests and coagglutination (Kaplan, L. K., et al., J. Clin. Micro., 14 (6):678-680, 1981; Ross, P. W. et al.: J. Clin. Pathol., 33:691-693, 1980; Cumming, C. G. et al., J. Med. Micro., 13:459-461, 1980; Facklam, R. R. et al., J. Clin. Micro., 15(6):987-990, 1982; Fung, J. et al., Am. J. Clin. Path., 5:608-610, 1982; and Edwards, E. A. et al., J. Clin. Micro., 15(3): 481-483, 1982).
Extraction of group-specific polysaccharides from the cell wall of streptococci is achieved with extraction reagents comprising an acid. The art teaches neutralization of acid extracted Group A Streptococcal antigen before carrying out an immunological assay procedure to detect the presence of the antigen. The neutralization step increases the time it takes to carry out such assays. It would therefore be advantageous to have an assay for acid extracted Group A Streptococcal antigen which is simpler and more rapid than those known in the art.
It would further be advantageous to have assay procedures for ligands extracted from cells or cell fragments which permit simultaneously carrying out the extraction of the ligand from the sample and reaction of the ligand with at least two anti-ligands therefor to form a detectable reaction product.